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Concentration ratios of morphine to codeine in blood of impaired drivers as evidence of heroin use and not medication with codeine.

Determining whether a person has taken heroin as opposed to a prescription drug containing codeine is not always easy because both opiates undergo metabolism into morphine (1-4). Moreover, clandestine heroin preparations contain acetylcodeine as an impurity, and this opiate is readily deacetylated to produce codeine (5, 6). Accordingly, forensic toxicology reports showing morphine and codeine in blood or urine could have several possible explanations. Heroin has a specific metabolite, 6-acetylmorphine (6-AM),[1] but its very short half-life of 10-20 min means that the window of detection in blood is only 1-2 h after administration (3, 4). For this reason, 6-AM is seldom identified in forensic blood samples above the limits of quantification (LOQs) by current methods of gas chromatography-mass spectrometry (GCMS) (7, 8). However, evidence of heroin intake is easier to obtain by analysis of urine specimens because 6-AM remains in urine for much longer than in blood (8-11).

The traffic police in Sweden try to obtain specimens of both blood and urine for toxicologic analysis, especially from individuals suspected of driving under the influence of drugs (DUID). This gave us the opportunity to compare the concentrations of morphine, codeine, and 6-AM in blood with the frequency of finding 6-AM in urine as unequivocal evidence of heroin intake.

Materials and Methods

All blood and urine specimens from impaired drivers apprehended in Sweden are submitted for analysis to one central laboratory, the National Laboratory of Forensic Chemistry located at the University Hospital in Linkoping. Drivers are apprehended for committing a moving traffic violation, during routine sobriety controls, or after traffic crashes. The material for this study was taken from the routine case files and toxicologic reports of blood or urine or both. Table 1 summarizes the numbers of DUID suspects involved in the study and whether blood or urine was available for analysis.

The police typically conduct a breath-alcohol screening test at the roadside with the aid of a hand-held instrument (12), either an Alcolmeter S-D2 or Alcolmeter 400 (Lion Laboratories). Performing various field-sobriety tests is unnecessary before making the preliminary breath-alcohol analysis. If the breath-alcohol test is negative and the police suspect impairment by drugs other than alcohol, specimens of blood and urine are taken for toxicologic analysis. The police make this decision on the basis of the general appearance and behavior of the suspect. More recently, the police also examine pupil size and eye movements, as well as other signs and symptoms of drug impairment. Examination of DUID suspects by drug recognition experts is not an option in Sweden. However, in some situations, a physician makes a clinical examination in conjunction with obtaining blood and urine samples for forensic toxicology.

Venous whole blood was submitted in two 10-mL gray-stoppered Vacutainer tubes (Becton Dickinson) containing 100 mg of sodium fluoride and 25 mg of sodium oxalate as preservatives. Urine specimens were sent for analysis in two 10-mL, plastic screw-capped tubes, each containing 100 mg of sodium fluoride as preservative. A qualitative screening analysis of urine and/or blood was made for five classes of abused drugs (opiates, cannabinoids, amphetamine analogs, cocaine metabolites, and benzodiazepines) by immunoassay techniques such as Emit or CEDIA with the Hitachi 717. The most commonly prescribed sedative-hypnotics and analgesics were analyzed in blood by capillary column gas chromatography with nitrogen-phosphorous detection (13). All positive immunoassay results were verified by more specific methods (liquid chromatography-mass spectrometry or GC-MS). After solid-phase extraction, opiates were determined by GC-MS with the use of deuterium-labeled internal standards and selected-ion monitoring (14). The blood aliquots were measured by weight and concentrations of drugs reported in mass/mass units (ng/g) because the fluidity of specimens differs widely, especially in postmortem bloods where it is not always practical to dispense aliquots by volume. After extraction and before gas chromatography, the opiates were converted to their pentafluoropropionic acid anhydride derivatives. For analysis of urine, the aliquots were measured by volume and results were thus reported mass/volume ([micro]g/L). Before GC-MS, the trimethylsilylester derivatives were prepared by treatment with N,O-bis(trimethylsilyl)trifluoroacetamide. The concentrations of opiates in blood and urine (morphine, codeine, and 6-AM) were quantitatively determined in a single GC-MS analytical run. The specimens were not hydrolyzed; therefore, the concentrations of unconjugated opiates could be reported. The LOQs for codeine, morphine, and 6-AM in blood were 5 ng/g and 20 [micro]g/L for urine. Calibration curves were constructed up to a maximum concentration of 1000 [micro]g/L in urine and values above this concentration were reported as >1000 [micro]g/L.

The frequency distributions of the concentrations of morphine and codeine in blood were markedly skewed, so the median value and 2.5 and 97.5 percentiles were used to characterize the data.

Results

Table 2 presents the drugs verified positive in blood from individuals apprehended for DUID in Sweden during 2000. Many individuals had several different drugs present in their blood, including scheduled narcotic drugs, prescription medications (mainly benzodiazepines), as well as various alcohol and drug combinations. Amphetamine and cannabis are the most commonly encountered drugs in DUID suspects in Sweden, but morphine and codeine are also high on the list of substances confirmed present in blood specimens.

Table 3 gives the concentrations of morphine, codeine, and 6-AM in blood samples from DUID suspects apprehended between 1992 and 2000. As expected, the concentrations span a wide range in this population, which comprises people taking therapeutic doses of opiates, as well as many drug addicts. The heroin metabolite 6-AM was determined 43 times at a median concentration of 8 ng/g (2.5 and 97.5 percentiles, 5 and 104 ng/g). The highly skewed distributions of morphine and codeine concentrations in blood are shown in Figs. 1 and 2 with medians of 30 ng/g for morphine and 20 ng/g for codeine.

In 675 opiate-positive blood samples, 6-AM was verified above the LOQ only 16 times (2.3%). By contrast, 6-AM was verified in 212 of 339 urine samples analyzed (62%). In 89 urine specimens containing 6-AM, the median morphine/ codeine ratio in the corresponding blood sample was 6.0 (range, 1-66). In 18 instances, 6-AM was present in urine, although morphine and codeine were below the LOQ in blood. Another 105 urine samples contained 6-AM when morphine was above the LOQ in blood (median concentration, 10 ng/g) but the concentration of codeine in blood was below the LOQ.

The frequency distribution of morphine/ codeine ratios in blood when urine was not available for analysis, and when morphine and codeine were both above the LOQ (n = 506), is shown in Fig. 3. In 76 instances (15%), the morphine/ codeine ratio was less than unity (median, 0.057), and the corresponding median blood-morphine concentration was 10 ng/g compared with 180 ng/g for codeine, which gives a median codeine /morphine ratio of 18:1. This high codeine /morphine ratio most likely reflects intake of the prescription drug codeine. In 430 cases, the morphine/ codeine ratio was greater than unity (median, 5.0; range, 1.1-67). For these cases, the median concentration of morphine was 70 ng/g (range, 7-620 ng/g) compared with 10 ng/g (range, 1-100 ng/g) for codeine, indicating heroin use. These results are summarized in Table 4.

Discussion

Since July 1999, Sweden has enforced a zero-tolerance law for DUID offenses, which means that the presence of a scheduled narcotic drug in blood is sufficient for prosecution regardless of whether the person exhibits any signs and symptoms of impairment. For the use of prescription drugs (e.g., benzodiazepines) without clear-cut evidence of impairment, the prosecution is required to prove that the recommended dose was exceeded. This requires an expert opinion to relate the blood drug concentration to the expected therapeutic concentration for the particular medication, which is not always easy.

[FIGURE 1 OMITTED]

If scheduled drugs like amphetamine, methamphetamine, cocaine, and tetrahydrocannabinol are present in blood above the LOQs by GC-MS, this is sufficient for prosecution, regardless of whether the persons driving ability was impaired. Zero-tolerance laws for narcotic drugs are similar in principle to the widely used per se laws for driving under the influence of alcohol. Because all specimens are sent to one forensic toxicology laboratory in Sweden (population 8.8 million), the LOQs for scheduled drugs in blood determined by GC-MS are effectively the threshold concentration limits for prosecution. Since the introduction of this zero-tolerance law for narcotics, the number of DUID cases submitted by the police has increased approximately fivefold. This sharp increase after July 1999 stems from an increased activity and enthusiasm by the police and prosecutors, who are now more likely to win a conviction. Moreover, the police are trained to recognize signs and symptoms of drug abuse and are allowed to monitor pupil size and look for nystagmus. This dramatic increase in the number of DUID cases can also be explained, at least in part, by prior knowledge about criminal elements and drug abusers in the community.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The ultimate evidence of heroin use is finding 6-AM in blood or urine. However, only 2.3% of opiate-positive DUID suspects were verified as heroin users based on the analysis of the 6-AM metabolite in blood. This compares with 62% confirmed users of heroin after the analysis of 6-AM in urine. The window of detection for 6-AM in blood and urine depends on the dose of heroin taken, the route of administration, and the frequency of heroin intake (10). The concentrations of opiates excreted in urine, including 6-AM, vastly exceeds the concentration in blood (15,16). Interpreting the concentrations of morphine and codeine measured in urine is more complicated, owing to the irregular frequency of urination and pooling of urine in the bladder, as well as variable diuresis and fluctuations in pH, which will affect the results of toxicologic analysis (16-19). Moreover, cleavage of glucuronides is often a tricky problem depending on whether acid hydrolysis or enzymatic methods are used and the source of [beta]-glucuronidase (20). The unconjugated drug concentrations of opiates in blood, more so than urine, reflect fairly recent intake (17).

Analysis of drugs of abuse in urine provides evidence of intake but does not permit conclusions to be drawn about coexisting concentrations in blood or impairment of the individual at the time of voiding. Even the concentrations of unconjugated opiates in blood or serum are difficult to relate to impairment, because of the development of tolerance. The introduction of so-called zero-tolerance laws for scheduled drugs in traffic cases sidesteps these problems and offers an effective means of combating the problem of DUID.

The forensic blood samples that are sent for analysis to our laboratory contain 1% sodium fluoride as preservative, which tends to cause hemolysis. Accordingly, the concentrations of drugs and poisons are determined in whole blood and not plasma or serum, which are the specimens more commonly used for therapeutic drug monitoring. Because the distribution ratio of morphine between plasma and whole blood is close to unity (21), finding a morphine/ codeine ratio >1.0 in plasma or serum also furnishes evidence of heroin use. Studies on the pharmacokinetics of codeine have shown that morphine/codeine ratios in plasma remain less than unity at all times after administration (1, 2,18). When 100 mg of codeine phosphate was given to healthy volunteers, the peak plasma morphine concentration was only 3.2% of the peak codeine concentration (19). Furthermore, the morphine/codeine ratios in plasma remained less than unity for up to 23 h postdosing (19). Similar results have been reported after a single oral dose of 60 mg of codeine and also after repeated intake (1,18). Morphine is also a metabolite of ethyl morphine, the active ingredient in various antitussive medications (22,23). However only a small fraction of the ethyl morphine is seemingly converted to morphine, and both parent drug and metabolite are easily detected in urine for up to 24 h after intake (22). Since the first report in Sweden in 1982 (24), the presence of various opiates in poppy seeds and other food products is well recognized as a confounding factor in urine drug testing programs. Finding 6-AM in urine can be used to dismiss the allegation that the source of morphine and codeine in urine was from foods laced with poppy seeds (25). The concentrations of unconjugated morphine and codeine in blood samples after eating poppy seed cakes will hardly be expected to exceed the LOQ of the GC-MS method of analysis (26).

Because of polymorphisms of the CYP2D6 enzyme, large interethnic differences in the rate and extent of demethylation of codeine exist (27). This leads to slow and rapid metabolizers of codeine depending on the particular genotype inherited (27, 28). Most Caucasians seem to be rapid metabolizers of codeine, and morphine/ codeine ratios within this population are always much less than unity (27). In a group of Chinese volunteers with low capability of metabolizing codeine, the morphine/ codeine concentration ratios after a dose of 50 mg of codeine were even less than in Caucasians. Accordingly, finding a high morphine/ codeine ratio in forensic blood specimens cannot be attributed to a person's inherent ability to convert codeine into morphine.

Many DUID suspects with morphine and codeine identified in blood claim they took the prescription drug codeine or received morphine for relief of pain (e.g., from injuries resulting from a traffic crash). However, the results reported here, based on finding 6-AM in urine, suggests that when morphine/ codeine ratios in blood are greater than unity, the person has more likely used heroin than taken the legal drug codeine. This allowed us to conclude that in 506 DUID suspects with morphine and codeine verified in blood above the LOQ, 85% had used heroin. Intake of mixtures of codeine and morphine or codeine and heroin obviously complicates the interpretation of morphine/ codeine ratios in blood. The rapid increase of immigration into Sweden has seen new drug cultures emerging. An example is the abuse of raw opium, which is likely to give a different pattern of morphine and codeine concentrations in body fluids. However, experience with two documented cases of raw opium abuse gave morphine/ codeine ratios in blood of 1.5 and 2.0. Accordingly, such cases would not be confused with the intake of codeine, for which morphine/ codeine ratios are always much less than unity (19).

From the results of this study, we recommend that if urine is not available for toxicologic analysis, the finding of a morphine/ codeine concentration ratio in blood exceeding unity is strong evidence that the person had used heroin, as opposed to having taken a prescription drug containing codeine.

References

(1.) Findlay JWA, Jones EC, Butz RF, Welch RM. Plasma codeine and morphine concentrations after therapeutic oral doses of codeine-containing analgesics. Clin Pharmacol Ther 1978;24:60-8.

(2.) Quiding H, Anderson P, Bondesson U, Boreus L-O, Hynning P-P,. Plasma concentrations of codeine and its metabolite morphine after single and repeated oral administration. Eur J Clin Pharmacol 1986;30:673-7.

(3.) Goldberger BA, Darwin WD, Grant TM, Allen AC, Caplan YH, Cone EJ. Measurement of heroin and its metabolites by isotope dilution electron-impact mass spectrometry. Clin Chem 1993;39:670-5.

(4.) Inturrisi CE, Max MB, Foley KM, Schultz M, Shin S-U, Houde RW. The pharmacokinetics of heroin in patients with chronic pain. N Engl J Med 1984;310:1213-7.

(5.) O'Neal CL, Poklis A. The detection of acetylcodeine and 6-acetylmorphine in opiate positive urines. Forensic Sci Int 1998;95:1-10.

(6.) Staub C, Marset M, Mino A, Mangin P. Detection of acetylcodeine in urine as an indicator of illicit heroin use: method validation and results of a pilot study. Clin Chem 2001;47:301-7.

(7.) Christophersen AS, Ceder G, Kristinsson J, Lillsunde P, Stenntoft A. Drugged driving in the Nordic countries-a comparative study between five countries. Forensic Sci Int 1999;106:173-90.

(8.) Jones AW. Heroin use by motorists in Sweden confirmed by analysis of 6-acetylmorphine in urine [Letter]. J Anal Toxicol 2001;25:353-5.

(9.) Fehn J, Megges G. Detection of 06-Monoacetylmorphine in urine samples by GC/MS as evidence for heroin use. J Anal Toxicol 1985;9:134-8.

(10.) Cone EJ, Welch P, Mitchell JM, Paul BD. Forensic drug testing for opiates: I. Detection of 6-acetylmorphine in urine as an indicator of recent heroin exposure; drug and assay considerations and detection times. J Anal Toxicol 1991;15:1-7.

(11.) Paul BD, Shimomura ET, Smith ML. A practical approach to determine cutoff concentrations for opiate testing with simultaneous detection of codeine, morphine, and 6-acetylmorphine in urine. Clin Chem 1999;45:510-9.

(12.) Jones AW. Evaluation of breath-alcohol instruments. IV. Roadside tests with Alcolmeter pocket model. Forensic Sci Int 1985;28: 157-65.

(13.) Eklund A, Jonsson J, Schuberth J. A procedure for simultaneous screening and quantification of basic drugs in liver, utilizing capillary gas chromatography and nitrogen sensitive detection. J Anal Toxicol 1983;7:24-8.

(14.) Schuberth J, Schuberth JS. Gas chromatographic-mass spectrometric determination of morphine, codeine and 6-monoacetyl morphine in blood extracted by solid phase. J Chromatogr 1989; 490:444-9.

(15.) Cone EJ, Welch P, Paul BD, Mitchell JM. Forensic drug testing for opiates, III. Urinary excretion rates of morphine and codeine following codeine administration. J Anal Toxicol 1991;15:161-6.

(16.) Lafolie P, Beck O, Lin Z, Albertioni F, Borous L. Urine and plasma pharmacokinetics of codeine in healthy volunteers: implications for drugs-of-abuse testing. J Anal Toxicol 1996;20:541-6.

(17.) Boemer U, Abbott S, Roe RL. The metabolism of morphine and heroin in man. Drug Metab Rev 1975;4:39-73.

(18.) Shah JC, Mason WD. Plasma codeine and morphine concentrations after a single oral dose of codeine phosphate. J Clin Pharmacol 1990;30:764-6.

(19.) Kronstrand R, Jones AW. Concentration ratios of codeine-tomorphine in plasma after a single oral dose (100 mg) of codeine phosphate [Letter]. J Anal Toxicol 2001;25:486-7.

(20.) Romberg RW, Lee L. Comparison of the hydrolysis rates of morphine-3-glucuronide and morphine-6-glucuronide with acid and 0-glucuronidase. J Anal Toxicol 1995;19:157-62.

(21.) Hand CW, Moore RA, Sear JW. Comparison of whole blood and plasma morphine. J Anal Toxicol 1988;12:234-5.

(22.) Popa C, Beck O, Brodin K. Morphine formation from ethylmorphine: implications for drugs-of-abuse testing in urine. J Anal Toxicol 1998;22:142-7.

(23.) Aasmundstad TA, Xu BQ, Johansson I, Ripel P,, Bjorneboe A, Christophersen AS, et al. Biotransformation and pharmacokinetics of ethylmorphine after a single oral dose. Br J Clin Pharmacol 1995;39:611-20.

(24.) Bjerver K, Jonsson J, Nilsson A, Schuberth J, Schuberth JS. Morphine intake from poppy seed food. J Pharm Pharmacol 1982;34:798-801.

(25.) Mule SJ, Casella GA. Rendering the "poppy-seed defense" defenseless: identification of 6-monoacetylmorphine in urine by gas chromatography/mass spectrometry. Clin Chem 1988;34:1427-30.

(26.) Hayes LW, Krasselt WG, Mueggler PA. Concentrations of morphine and codeine in serum and urine after ingestion of poppy seeds. Clin Chem 1987;33:806-8.

(27.) Yue QY, Hasselstr6m J, Svensson JO, Sawe J. Pharmacokinetics of codeine and its metabolites in Caucasian healthy volunteers: comparisons between extensive and poor hydroxylators of debrisoquine. Br J Clin Pharmacol 1991;31:635-42.

(28.) Yue QY, Svensson JO, Sjogvist F, Sawe J. A comparison of the pharmacokinetics of codeine and its metabolites in healthy Chinese and Caucasian extensive hydroxylators of debrisiquine. Br J Clin Pharmacol 1991;31:643-7.

GUNNEL CEDER AND ALAN WAYNE JONES *

Department of Forensic Chemistry, University Hospital, 58185 Linkaping, Sweden.

[1] Nonstandard abbreviations: 6-AM, 6-acetylmorphine; LOQ, limit of quantification; GC-MS, gas chromatography mass spectrometry; and DUID, driving under the influence of drugs.

* Author for correspondence. Fax 46-13-104875; e-mail wayne.jones@RMV.SE.

Received May 8, 2001; accepted August 7, 2001.
Table 1. Distribution of DUID cases in Sweden before and
after introduction of zero-tolerance legislation (July 1999).

Specimens available January 1992 July 1999 to
for analysis to June 1999 December 2000

Blood only 4569 (77%) 2922 (56%)
Both blood and urine 1348 (23%) 2248 (44%)
Total 5917 (100%) 5176 (100%)

Table 2. Analytical results for the most commonly encountered drugs in
blood samples from DUID suspects in Sweden during 2000. (a)

Drug Year 2000 (n = 3808)

Amphetamine 2395 (63%)
Tetrahydrocannabinol 1119 (29%)
Diazepam 743 (20%)
Morphine 382 (10%)
Methamphetamine 364 (10%)
Flunitrazepam 347 (9%)
Codeine 245 (6%)
Paracetamol (acetaminophen) 129 (3%)
MDMA 116 (3%)
Phenazone 109 (3%)
Benzoylecgonine 89 (2%)
Nitrazepam 81 (2%)
Alprazolam 80 (2%)
d-Proxyphene 67 (2%)

(a) Note that many specimens contained several different drug classes.

Table 3. Concentrations of morphine, codeine, and 6-AM in
opiate-positive blood specimens from motorists apprehended
for DUID in Sweden between 1992 and 2000.

 Concentration of opiates
 in blood, median (2.5
Opiate Number and 97.5 percentiles)

Morphine 979 30 ng/g (5 and 230)
Codeine 784 20 ng/g (5 and 592)
6-AM 43 8 ng/g (5 and 104)

Table 4. Concentrations of morphine and codeine in blood
samples from motorists apprehended for DUID in Sweden
when both opiates were above the LOQs (5 ng/g)

 Median (2.5 and
Opiate in blood Number 97.5 percentiles)

Morphine 506 50 ng/g (6 and 268)
Codeine 506 10 ng/g (5 and 451)
Morphine/Codeine ratio >1 430 Morphine, 70 ng/g;
 codeine, 10 ng/g
Morphine/Codeine ratio <1 76 Morphine, 10 ng/g;
 codeine, 180 ng/g
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Title Annotation:Drug Monitoring and Toxicology
Author:Ceder, Gunnel; Jones, Alan Wayne
Publication:Clinical Chemistry
Date:Nov 1, 2001
Words:3545
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